WO1998001755A9 - Procede de criblage de proteines mena intervenant dans la dynamique des microfilaments - Google Patents

Procede de criblage de proteines mena intervenant dans la dynamique des microfilaments

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WO1998001755A9
WO1998001755A9 PCT/US1997/011669 US9711669W WO9801755A9 WO 1998001755 A9 WO1998001755 A9 WO 1998001755A9 US 9711669 W US9711669 W US 9711669W WO 9801755 A9 WO9801755 A9 WO 9801755A9
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mena
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  • the invention relates to DNA seguences encoding proteins involved in microfilament dynamics. More specifically, the invention relates to DNA sequences and proteins involved in microfilament dynamics affecting normal and abnormal cell morphology, adhesion, motility, growth and differentiation, as well as pathogenicity of certain viruses and bacteria, and to screening, diagnostic and therapeutic methods and compositions employing these DNA sequences and proteins.
  • the control of cell morphology, motility, growth and differentiation generally requires coupling of external stimuli to processes that regulate cytoskeletal architecture.
  • the mechanical forces that drive morphological change and migration arise initially from the microfilament-based cytoskeleton (reviewed by Lauffenburger and Horwitz, Cell 84: 359-369, 1996; Mitchison and Cramer, Cell 84: 371-379, 1996).
  • formation of cellular protrusions such as filopodia and lammellipodia requires polymerization and stabilization of F-actin.
  • Actin-driven formation of membrane protrusions is the first step in migration of neuronal growth cones (Forscher et al . , Nature 357:, 515-8, 1992; Forscher and Smith, S., J “ . Cell Biol . 107:1505-1516, 1988; Sanders and Wang, J “ . Cell Sci . 100:771-780 , 1991).
  • Establishment of proper connections in the central nervous system depends on the ability of neuronal growth cones to guide neurites to their final targets.
  • Dab Disabled
  • CNS embryonic central nervous system
  • Ena protein is concentrated in CNS axons, like Abl and Dab, and has a proline-rich core which binds in vi tro to the SH3 domains of Abl and Src, but not to the SH3 domains of several other proteins tested (Gertler et al . , Genes Dev. 9:521-33, 1995).
  • Ena is tyrosine phosphorylated in vivo. The phospho yrosine content of Ena is reduced approximately three- fold in Abl mutant pupae, indicating that Ena is phosphorylated by Abl -dependent and independent tyrosine kinases. Over expression and in vitro phosphorylation studies demonstrate that Abl can phosphorylate Ena directly.
  • Listeria motility and intercellular infection depends on the asymmetric recruitment of host profilin, an actin monomer binding protein, to one pole of the bacteria where rapid polymerization of actin is induced to form a microfilamentous "comet tail" that drives bacterial movement (Theriot et al . , Cell 76:505-517, 1994).
  • VASP Vasodilator-Stimulated Phosphoprotein
  • VASP is distributed along microfilaments, is particularly concentrated at sites of focal contact and in the peripheral lammelli of spreading or migrating cells, and there is evidence of direct contact between VASP and F-actin (Reinhard et al . , EMBO J. 11:2063-2070, 1992; Haffner et al . , EMBO J . 14:19-27, 1995).
  • VASP also contains a central region of proline-rich sequences comprising a proposed binding domain for the actin binding protein profilin (Reinhard et al . , EMBO J. 14:1583-1589, 1995) .
  • VASP is recruited to the bacterial surface by direct binding with the ActA surface protein (Chakraborty et al . , EMBO J. 14:1314-1321, 1995; Pistor et al . , Curr. Biol. 5:517-525, 1995). VASP accumulates on the bacterial surface prior to the formation of F-actin clouds that precede comet tail formation, and is later localized at the site of actin filament assembly between the polar bacterial surface and the front of the actin tail (Chakraborty et al . , EMBO J. 14:1314-1321, 1995). VASP is also a ligand for profilin (Reinhard et al . , EMBO J. 14:1583-1589, 1995).
  • the invention achieves these objects and other objects and advantages which will become apparent from the description which follows by providing two novel mammalian genes Mammalian Ena (Mena) , and Ena -VASP like (Evl) encoding novel proteins Mena, and Evl .
  • the Mena and Evl proteins provided herein possess novel biochemical and cell biological properties rendering these proteins useful in the study and/or control of a variety of microfilament dynamic processes.
  • the Mena and Evl proteins disclosed herein are useful for generating, isolating, and characterizing additional endogenous and exogenous factors, drugs and other agents useful for evaluating and/or controlling cytoskeletal dynamics involved in normal and abnormal cell morphology, adhesion, motility, growth and/or differentiation. Reflective of these uses, the invention provides several methods and tools specifically directed to the diagnosis and treatment of injury and disease conditions involving cytoskeletal dynamic processes, such as metastatic cancer and infection by cytopathogenic bacteria.
  • purified and isolated polynucleotides which encode a native Mena or Evl protein.
  • these polynucleotides are DNA sequences encoding specific native isoforms of Mena, including an approximately 140 kD, neural-specific isoform of Mena, an approximately 60 kD hematopoietic-specific Mena isoform, and approximately 80 kD and 88 kD isoforms of Mena which are broadly distributed among different mammalian tissue types.
  • Mena or Evl based polynucleotides that encode Mena or Evl proteins or fragments thereof, recombinantly produced peptide derivatives, fusion proteins and the like incorporating only a portion of Mena or Evl , and up to the entire Mena or Evl protein.
  • Mena or Evl based polynucleotides are sequences that encode discrete functional domains of the Aena or Evl protein responsible for specific biological activities of the intact protein.
  • Preferred functional domains in this context include an N-terminal domain of Mena and Evl that mediates binding of Afena to certain proline-rich ligands, including ActA, zyxin and vinculin, and which directs localization of Mena and heterologous proteins linked to the EVHl domain to focal contacts, and to the surface of cytopathogenic bacteria. Also provided are mutagenized polynucleotides that encode mutant analogs of the Mena or Evl protein, or of selected polypeptide portions of Mena or Evl such as the EVHl functional domain, which exhibit modified biological activity compared to native Mena or Evl proteins and polypeptide domains.
  • substantially pure Mena or Evl proteins are provided, including a variety of tissue specific or broadly distributed Mena and Evl isoforms disclosed herein. Also provided are Mena or Evl based synthetic peptides or fragments thereof, recombinantly produced peptides, fusion proteins and the like incorporating all or a portion of Mena or Evl , including peptides corresponding to the aforementioned Mena or Evl functional domains, as well as mutant analogs of the Mena or Evl protein or polypeptide portions thereof.
  • antibodies, antibody derivatives and chimeric antibodies are provided that bind to one or more of the Mena and Evl proteins and isoforms thereof. Also provided are antibodies, antibody derivatives and chimeric antibodies that bind to one or more of the Mena or Evl based synthetic peptides, recombinantly produced peptides, fusion proteins and the like disclosed herein.
  • Mena or Evl based recombinant polynucleotide expression constructs which include a polynucleotide insert encoding a Mena or Evl protein or Mena or Ena based synthetic peptide, recombinantly derived peptide, fusion protein or the like operably incorporated within an expression vector capable of directing expression of the polynucleotide insert sequence in a mammalian host cell.
  • Various such constructs are provided, including constructs that incorporate polynucleotide sequences encoding the native 140 kD, 60 kD, 80 Kd or 88 Kd Mena isoforms.
  • Mena or Evl based expression constructs are employed in methods for modulating expression of Mena or Evl , disrupting Mena or Evl expression or inducing ectopic expression of Mena or Evl genes and related polynucleotide sequences in mammalian host cells.
  • the expression constructs are introduced into the host cells by microinjection, transfection or other suitable method to achieve transformation of the host cell, and the host cell is cultured, transplanted or left in si tu under suitable conditions to allow the host cell to express the Mena or Evl based polynucleotide insert sequence.
  • the invention provides screening, diagnostic and therapeutic methods that utilize Mena or Evl encoding polynucleotides, Mena or Evl proteins, peptides, fusion proteins and the like, Mena or Evl binding antibodies, Mena or Evl based expression constructs, and/or mammalian host cells transformed to express Mena or Evl insert sequences.
  • labeled Mena or Evl proteins and peptides are used to determine the localization and activity of Mena or Evl in normal and abnormal cytoskeletal dynamic processes.
  • labeled Mena proteins, peptides and antibodies are introduced into the cytoplasm of Listeria infected cells to detect and quantify recruitment of Mena to the bacterial surface at the polar site of comet tail formation, and to detect and quantify the activity of Mena in mediating Listeria induction of microfilament polymerization.
  • Mena and anti-Mena antibodies are used to detect and quantify Mena binding to ActA, profilin, zyxin or vinculin.
  • Each of these methods in turn provide a basis for additional methods to screen for agonists and antagonists of specific Mena activities involved in the Listeria microfilament induction pathway.
  • Evl proteins and peptides are used to detect and quantify Mena or Evl activity in normal and abnormal cytoskeletal dynamic processes under endogenous control.
  • Mena or Evl activity is detected and quantify Mena or Evl activity in normal and abnormal cytoskeletal dynamic processes under endogenous control.
  • Listeria microfilament induction model localization and quantification of Mena or
  • Evl associated with dynamic cytoskeletal structures or events, or with specific molecular factors involved in cytoskeletal regulation provides the basis for a variety of methods to diagnose abnormal cytoskeletal processes associated with disease, and to screen for agonists and antagonists of specific Mena activities involved in such processes.
  • labeled Mena protein is used as a reagent to screen small molecule and peptide libraries to identify inhibitors of Mena activity.
  • an overlapping set of chemically synthesized peptides is generated covering the sequence of a selected Mena ligand, such as ActA, Profilin or Zyxin, and this library is screened using covalently or antibody labeled Mena to identify highly specific and potent peptide inhibitors.
  • Mena ligand such as ActA, Profilin or Zyxin
  • Mena or Evl based synthetic peptides, recombinantly produced peptides, fusion proteins and the like incorporating all or a portion of Mena or Evl , as well as mutant analogs of Mena or Evl proteins or peptides, are used to screen for specific agonists and antagonists of Mena or Evl activities.
  • the EVHl domain of Mena or Evl , or an EVHl consensus peptide having a conserved amino acid sequence between Mena, Evl and VASP is used as an affinity reagent to identify as yet unknown cellular factors involved in cytoskeletal regulation, or as a labeled probe to screen cDNA expression libraries to identify genes encoding such unknown regulatory factors.
  • Other screening and diagnostic methods of the invention rely on labeled polynucleotide probes to map the chromosomal locations of Mena, Evl and related genes, to identify genetic defects in these genes among cell populations or individuals, and to detect and measure expression of Mena, Evl or related genes in association with specific cytoskeletal dynamic structures, events or molecular regulatory factors.
  • the invention provides a range of therapeutic methods for preventing and/or treating diseases and other conditions that involve aberrant cytoskeletal regulation, for example pathogenic infection, traumatic neural injury and cancer.
  • Therapeutic methods of the invention variously utilize Mena or Evl encoding polynucleotides, Mena or Evl proteins and peptides, Mena or Evl binding antibodies, Mena or Evl based expression constructs, mammalian host cells transformed to express Mena or Evl insert sequences, and other therapeutic agents such as triplex forming oligonucleotides, antisense polynucleotides or ribozymes that specifically target Mena and/or Evl polynucleotides.
  • agents that inhibit Mena or Evl expression or activity and thereby eliminate or impair aberrant recruitment of Mena by pathogens, or interrupt Mena expression or activity associated with cancerous cell migration.
  • Useful inhibitors in this context include antibodies that block or impair Mena or Evl by binding to the protein, Mena or Evl based, synthetic or recombinantly produced peptides, fusion proteins that compete for Mena or Evl binding partners or otherwise block or impair Mena or Evl expression or activity, triplex forming oligonucleotides, antisense polynucleotides or ribozymes that block expression of Mena and/or Evl polynucleotides, and mutant Mena or Evl based expression constructs that abolish or reduce Mena or Evl expression or activity in targeted cells.
  • agents that induce or enhance Mena or Evl expression or activity include anti- Evl antibodies, Mena or Evl based, synthetic or recombinantly produced peptides, fusion proteins and the like that enhance Mena or Evl binding to its partners or otherwise enhance Mena or Evl expression or activity, and mutant Metia or Evl based expression constructs that induce or enhance Mena or Evl expression or activity in targeted cells.
  • the present invention provides two novel mammalian genes Mammalian Ena (Mena) , and Etia -VASP like (Evl) which encode novel proteins Mena and Evl .
  • Sequences encoding Mena and Evl include those sequences that result in minor variations in amino acid sequence, such as those due to genetic polymorphisms, allelic variations, differences between species and those in which blocks of amino acids have been added, altered or replaced without substantially altering the biological activity of the proteins.
  • Mena and Evl genes and Mena and Evl proteins disclosed herein are useful for generating, isolating, and characterizing endogenous and exogenous factors, drugs and other agents that can be employed in methods to evaluate and/or regulate cytoskeletal dynamic processes involved in normal and abnormal cell morphology, adhesion, motility, growth and/or differentiation.
  • the invention provides specific methods directed toward the diagnosis and treatment of injury and disease conditions involving cytoskeletal dynamic processes, such as metastatic cancer and infection by cytopathogenic bacteria.
  • purified and isolated polynucleotides which encode a native Mena or Evl protein.
  • these polynucleotides are DNA sequences encoding specific isoforms of native Mena or Evl .
  • Preferred methods to isolate and purify polynucleotides encoding native Mena or Evl employ conventional screening, subcloning and polymerase chain reactions, for example according to the methods and using the reagents described in Sambrook et al . , (1989) Molecular Cloning A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Press) .
  • a restriction fragment of the Drosophila gene Ena (Gertler et al . , Genes Dev.
  • Additional polynucleotide probes that are useful within the invention include Mena or Evl probes corresponding to partial or complete nucleotide sequences generated from the cDNAs depicted in SEQ ID. NOS. 1, 3 or 7 below. Degenerate oligonucleotides based on the amino acid sequences of SEQ ID NOS. 2, 4, 5, 6, 8 and 9 are also useful in methods for identifying additional isoforms and related sequences.
  • proteins, synthetic peptides, recombinantly produced peptides, fusion proteins and the like that bind or otherwise interact with Mena can be used as labeled probes to screen mammalian cDNA expression libraries to identify additional genes or cDNAs encoding Mena or Evl proteins .
  • known Mena binding partners including ActA, zyxin, vinculin and VASP, are used as probes to detect Mena encoding polynucleotides, for example using well known autoradiographic or immunoassay screening methods.
  • hybridization conditions will be evident to one skilled in the art and will generally be guided by the purpose of the hybridization, the type of hybridization (DNADNA or DNA-RNA) , and the level of relatedness between the sequences.
  • Methods for hybridization are well established in the literature; see, for example: Sambrook, ibid. ; Hames and Higgins, eds, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington DC, 1985; Berger and Kimmel, eds, Methods in Enzymology, Vol.
  • Hybridization stringency can be altered by adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and salt concentration of the wash solutions.
  • the stringency of hybridization is adjusted during the post- hybridization washes by varying the salt concentration and/or the temperature.
  • Stringency of hybridization may be reduced by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution.
  • High stringency conditions may involve high temperature hybridization (e.g., 65-68°C in aqueous solution containing 4-6XSSC, or 42 °C in 50% formamide) combined with high temperature (e.g., 5-25°C below the T m ) and a low salt concentration (e.g., 0. IX SSC).
  • Reduced stringency conditions may involve lower hybridization temperatures (e.g., 35-42 °C in 20-50% formamide) with intermediate temperature (e.g., 40-60°C) and washes in a higher salt concentration (e.g., 2-6X SSC).
  • Moderate stringency conditions which may involve hybridization at a temperature between 50 °C and 55 °C and washes in 0.
  • Ix SSC 0.1% SDS at between 50 °C and 55 °C, may be used to identify clones encoding polynucleotide molecules encoding Mena or Evl from other species or to isolate isoforms of Mena or Evl .
  • Mena or Ena encoding polynucleotides that are particularly useful within the invention encode Mena or Ena proteins having unique structural and/or biochemical properties, or which exhibit discrete expression or tissue distribution patterns among various Mena or Ena isoforms.
  • SEQ ID NO. 1 depicts a Mena cDNA polynucleotide and is deduced amino acid sequence (SEQ ID NOS.
  • Neural Mena cDNA polynucleotides that contain combinations of three alternately included exons and encoding multiple isoforms of Mena (Neural Mena + , SEQ ID NO. 4; Neural Mena ++ , SEQ ID NO. 5; and Neural Mena +++ , SEQ ID NO. 6) .
  • a cDNA encoding Neural Mena is disclosed (SEQ ID NO. 3) .
  • Neural Mena isoforms are approximately 140 kD in size and exhibit neural tissue specific distribution.
  • cDNA polynucleotides encoding an approximately 60 kD, hematopoietic specific form of Mena, and cDNA polynucleotides (SEQ ID NOS. 7) encoding the Evl protein (SEQ ID NO. 8) are described.
  • SEQ ID NOS. 7 cDNA polynucleotides encoding the Evl protein
  • molecular size of proteins translated from Mena or Ena coding cDNAs in vi tro can be determined by gel electrophoresis, and tissue distribution can be determined by comparative Western blotting of cell lysates from different tissues, or in si tu hybridization of whole embryos or tissue sections, using anti -Mena or anti-.Evl antibodies (for example according to the general methods disclosed in Harlow and Lane (1988) ; Antibodies A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Press) , which is incorporated herein by reference in its entirety) .
  • tissue specificity of Mena or Ena encoding polynucleotides include assays that measure Mena or Ena expression between tissue differentiation stages, for example in in vivo developmental models or systems where tissue differentiation can be induced by exogenous stimuli.
  • Mena protein is assayed by direct anti-Me ⁇ a immunofluorescence assay in developing embryos and in differentiated P19 embryonic carcinomal cells, to demonstrate that expression of the 140 kD neural specific Mena isoform increases during development at the time of rapid neurite outgrowth, and that subcellular distribution of Neural Mena in P19 cells is concentrated in the growth cones of developing neurites.
  • Mena or Evl based polynucleotides that encode Mena or Evl proteins or fragments thereof, recombinantly produced peptide derivatives, fusion proteins and the like incorporating only a portion of Mena or Evl , and up to the entire Mena or Evl protein.
  • Mena or Evl based polynucleotides are sequences that encode discrete functional domains of the Mena or Evl protein responsible for specific biological activities of the intact protein.
  • N-Mena N-terminal domain of Mena (N-Mena) including approximately the first 170 amino acids of the protein is demonstrated to mediate binding of Mena to certain proline-rich ligands, including ActA, zyxin and vinculin, and to direct localization of Mena and heterologous proteins linked to the N-Mena peptide to focal contacts, and to the surface of cytopathogenic bacteria.
  • the N-Mena peptide corresponds to a functional domain (the EVHl domain) that is highly conserved between Mena, Evl and VASP, as demonstrated by sequence alignment comparisons. A consensus sequence for the alignment is shown in SEQ ID NO. 9.
  • substantially pure Mena or Evl proteins and peptides are provided that are initially encoded by the aforementioned polynucleotides.
  • the Mena and Evl proteins include a variety of tissue specific or broadly distributed Mena and Evl isoforms disclosed herein.
  • Mena or Evl based synthetic peptides, recombinantly produced peptides, fusion proteins and the like incorporating all or a portion of Mena or Evl , including peptides corresponding to the aforementioned Mena or Evl functional domains, as well as mutant analogs of the Mena or Evl protein or polypeptide portions thereof.
  • proteins or polypeptide portions thereof may or may not have the biological activity of corresponding native Mena or Evl .
  • proteins are prepared that are capable of binding to the proline-rich binding partner ActA, but not capable of inducing profilin.
  • the peptides can be prepared via chemical synthesis, as described hereinbelow, or by recombinant DNA technology, or as fusion proteins, and the like. Desirably, the peptide will be as small as possible while still maintaining substantially all of the reactivity of a larger peptide.
  • the subject peptides have an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions with an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NOS.
  • amino acids 6-170 amino acids 6-170
  • amino acids 440-537 amino acids 440-537
  • SEQ ID NO. 11 amino acids 6-170
  • amino acids 440-537 amino acids 440-537
  • SEQ ID NO. 11 amino acids 6-170
  • C-Mena amino acids 440-537
  • SEQ ID NO. 11 amino acids 6-170
  • SEQ ID NO. 11 amino acids 440-537
  • SEQ ID NO. 11 amino acids 6-170
  • C-Mena amino acids 440-537
  • SEQ ID NO. 11 amino acids 6-170
  • C-Mena amino acids 440-537
  • the peptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or nonconservative, where such changes might provide for certain advantages in their use.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the sequence of the peptide will not differ by more than about 20% from the native Mena or Evl sequence, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, e.g., ease of linking or coupling, and the like.
  • additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, e.g., ease of linking or coupling, and the like.
  • the peptides in the composition can be identical or different, and together they should provide equivalent or greater reactivity than the parent peptide (s) .
  • the subject peptides find a variety of uses including preparation of specific antibodies.
  • the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis , 2d. ed. , Pierce Chemical Co. (1984); Tam et al . , J. Am . Chem . Soc . 105:6442 (1983); Merrifield, Science 232:341-347 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284 (1979), each of which is incorporated herein by reference.
  • Short peptide sequences or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • PCR is used to generate polynucleotide fragments encoding amino acids corresponding to a selected or putative functional domain, for example amino acids 6-170 representing N-Mena, or amino acids 440-537 representing a C-terminal functional domain of Mena (C-Mena) .
  • the polynucleotide fragments are then cloned into a suitable expression vector, for example pGex2T, and fusion proteins are prepared (Smith and Johnson, Gene 67:31-40, 1988, which is incorporated herein by reference in its entirety) .
  • the fusion proteins can be endogenously labeled, for example using a covalent radiolabel or other conventional marker, or alternately labeled using antibodies that bind to the fusion protein.
  • an N-Mena-Gst fusion protein was generated and immobilized on glutathione-agarose bead matrix (SEPHAROSE; Pharmacia Biotech, Inc., Piscataway, NJ) . Proteins capable of binding N-Mena-Gst were then purified from head lysates of stage E12 mouse embryos, or human erythrocytic leukemia cells (HEL) . Bound proteins were analyzed by Western blotting with antibodies to ActA, zyxin, vinculin or the Ack kinase.
  • N-Mena binds to ActA, zyxin and vinculin, but not to Ack. More specific examples, employing ligand overlay assays using peptides derived from vinculin or ActA (for example according to the methods of Chakraborty et al . EMBO J. 14:1314-1321, 1995) demonstrate that N-Mena can associate with motifs common to ActA, zyxin and vinculin.
  • N-Mena and other functional domains of Mena and/or Evl in cytoskeletal dynamic processes can be further evaluated and incorporated within the methods of the invention using a variety of additional tools that are disclosed herein, or are elsewhere described and known in the art.
  • purified Gst-N-Mena is icroinjected into PtK2 cells seeded to semi- confluency on CELLocate coverslips (Eppendorf , Hamburg, Germany) , and anti-Gst antibody assays coupled with phalloidin labeling to show the distribution of microfilaments is used to demonstrate that the injected fusion protein localizes to focal adhesions, whereas Gst injected alone is diffusely localized throughout the cytoplasm.
  • a peptide containing the ActA sequence SEPSSFEFPPPPTDEELRLA (SEQ ID NO. 12) is injected into the cells in an attempt to saturate the N-Mena binding site for ActA.
  • SEPSSFEFPPPPTDEELRLA SEQ ID NO. 12
  • Gst-N-Mena is observed to be depleted from the focal contacts, whereas distribution of the fusion protein is unaffected by injection of an unrelated ActA peptide.
  • Similar competition assays demonstrate depletion of endogenous Mena in ActA peptide injected cells.
  • N-Mena and other functional domains of Mena and/or Evl in cytoskeletal dynamic processes can be further evaluated and incorporated within the methods of the invention in the context of pathogenic induction of cytoskeletal dynamic processes.
  • purified Gst-N-Mena was microinjected into PtK 2 cells which were then infected with Listeria .
  • Anti-Gst immunoassay coupled with phalloidin labeling shows that the Mena based fusion protein associates with the surface of the bacteria and overlaps with actin in the region of actin polymerization at the tail of the bacterium, similar to the distribution of endogenous Mena in Listeria infected cells.
  • Mena protein translated in vi tro binds immobilized profilin, which binding is reduced by six- fold following preincubation of the profilin matrix with a (GP 5 ) 3 peptide (SEQ ID NO. 12) corresponding to the profilin-binding motif of VASP (Reinhard et al . , EMBO J. 14:1583-1589, 1995, which is incorporated herein by reference in its entirety) .
  • GP 5 GP 5 3 peptide
  • VASP profilin-binding motif of VASP
  • the general methods and compositions described above for localizing and quantifying Mena or Evl associated with dynamic cytoskeletal structures or events, and for determining Mena activity in association with specific factors and events involved in cytoskeletal regulation provides the basis for a variety of methods to diagnose and treat abnormal cytoskeletal processes associated with pathogenic infection, disease and other conditions, and to screen for agonists and antagonists of specific Mena activities involved in such processes.
  • a particularly useful set of tools in this context includes the various antibodies provided within the invention for detecting Mena and/or Evl expression, activity and localization.
  • Antibody reagents useful for these purposes include antibodies, antibody derivatives and chimeric antibodies that bind to one or more of the Mena and Evl proteins and isoforms thereof. Also provided are antibodies, antibody derivatives and chimeric antibodies that bind to one or more of the Mena or Evl based synthetic peptides, recombinantly produced peptides, fusion proteins and the like disclosed herein.
  • non-human antisera or monoclonal antibodies e.g., murine, lagomorpha, porcine, equine
  • general methods for the production of non-human antisera or monoclonal antibodies are well known and may be accomplished by, for example, immunizing an animal with Mena protein or Mena based peptides or alternatively with Evl protein or Evl based peptides.
  • antibody producing cells are obtained from immunized animals, immortalized and screened, or screened first for the production of an antibody that binds to the Mena protein or peptides and then immortalized.
  • a human monoclonal antibody or portions thereof may be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to Mena according to the method generally set forth by Huse et al . ( Science 246:1275-1281, 1989, which is incorporated by reference herein in its entirety) . The DNA molecule may then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity.
  • Anti-Mena or anti- Evl antibodies are particularly useful within the invention as labeled reagents to detect, image and/or quantify the presence or activity of Mena or Evl , or of agonists or antagonists of Mena or Evl activity.
  • anti-Mena and anti-EV1 antibodies are provided which incorporate one or more well known labels such as a dye, fluorescent tag or radiolabel.
  • Anti-Mena or anti-EVl antibodies may also be used themselves as agonists or antagonists of Mena or Evl activity, for example by interfering with the binding of Mena to ActA, profilin, zyxin or vinculin.
  • anti-Mena or anti-Bvl antibodies may be used as targeting agents for the delivery of compounds of therapeutic interest.
  • Such compounds include, but are not limited to, toxins, cytostatic compounds, or proenzymes whose potential function is to activate endogenous proenzymes, to activate proenzymes from exogenous sources, or to activate enzyme cleavage sites on prodrugs.
  • toxins include, but are not limited to, toxins, cytostatic compounds, or proenzymes whose potential function is to activate endogenous proenzymes, to activate proenzymes from exogenous sources, or to activate enzyme cleavage sites on prodrugs.
  • proenzymes whose potential function is to activate endogenous proenzymes, to activate proenzymes from exogenous sources, or to activate enzyme cleavage sites on prodrugs.
  • bifunctional antibodies having independent antigen binding sites on each immunoglobulin molecule (as disclosed for example in Thromb . Res . Suppl . X:83, 1990, and in T!he Second Annual IBC International Conference on Antibody Engineering, A. George ed. , Dec. 16-18, 1991
  • Bifunctional antibodies and antibody panels of particular use within the invention include antibodies and panels of antibodies that bind to both Mena and Evl , or to multiple functional domains of the Mena or Evl protein.
  • the subject proteins and peptides of the invention are also useful as reagents to detect, image and/or quantify the presence or activity of Mena or Evl , or of agonists or antagonists of Mena or Evl activity, in addition to their usefulness in the preparation of anti-Mena and anti-EvI antibodies.
  • a collection of Mena or Evl proteins substantially purified are provided, including all of the Mena and Evl isoforms disclosed herein.
  • Mena and Evl proteins produced according to the present invention may be purified using a number of established methods such as affinity chromatography using anti-Mena or anti-Evl antibodies coupled to a solid support. Additional purification may be achieved using conventional purification means such as liquid chromatography, gradient centrifugation and gel electrophoresis among others. Methods of protein purification are known in the art (see generally, Scopes, R. , Protein Purification, Springer-Verlag, NY, 1982, which is incorporated herein by reference) and may be applied to the purification of recombinant Mena or Evl described herein. Thus, Mena and Evl are provided isolated from its natural cellular environment, and substantially free of other cellular proteins.
  • Purified Mena and Evl are provided, where substantially pure Mena and Evl of at least about 50% is preferred, at least about 70-80% is more preferred, and 95-99% or more homogeneity most preferred.
  • the recombinant Mena and Evl or native Mena and Evl may be used to generate antibodies, diagnostically in assay procedures, etc.
  • Proteins, peptides and fusion proteins of the invention may be selected to include one or more of the Mena or Evl functional domains disclosed herein, as well as mutant analogs of the Mena or Evl protein or polypeptide which exhibit modified biological activity compared to native Mena or Evl proteins and polypeptide domains.
  • the proteins, peptides and fusion proteins of the invention are provided according to the methods disclosed herein, as well as by additional recombinant, biosynthetic and peptide chemical methods described elsewhere and generally known in the art.
  • site directed mutagenic techniques that can be used to generate targeted mutations in specific regions or functional domains of a Mena or Evl based polynucleotide.
  • site directed mutagenesis within the EVHl domain encoding portion of Mena will yield Mena mutants that are either hyperfunctional or dominant negative with respect to Mena activities attributable to the EVHl domain, including ActA, zyxin and vinculin binding activities, and subcellular localization of Mena .
  • Such mutants can be useful by themselves in various applications, or combined with other native or mutant peptide sequences, labels or other agents for more specific purposes.
  • an N-Mena mutant having an enhanced ActA, zyxin or vinculin binding activity selective for a specific binding partner can be used to screen for particularly strong or specific antagonists that inhibit that specific binding activity.
  • hyperfunctional N-Mena mutants will be particularly useful as gene therapeutic agent to induce or enhance Mena function in impaired systems, such as in the case of a neural injury or wound healing condition.
  • hyperfunctional N-Mena mutants can be operably joined to other peptide sequences, labels or chemical agents to achieve a particular therapeutic value.
  • One such chimeric mutant is contemplated having a hyperfunctional N-Mena mutation as well as a dominant negative mutation in the proline rich, profilin binding domain.
  • This particular chimera will compete strongly with endogenous Mena for ActA, zyxin and vinculin binding, but will fail to promote wild type profilin binding and microfilament assembly.
  • Such a mutant will be useful tool in a variety of applications, for example to screen for agonists that replace or enhance Mena-profilin binding activity, or as a gene therapeutic agent capable of inhibiting endogenous Mena function in aberrant migratory cells, such as Chronic Myelogenous Leukemia (CML) and other cancer cells.
  • CML Chronic Myelogenous Leukemia
  • Mena or Evl proteins, peptides, fusion proteins and antibodies are used in a variety of screening and diagnostic methods.
  • the polynucleotide molecules, protein, peptides and antibodies of the present invention are useful in in vi tro assays to screen for compounds capable of modulating the activity of expression of Mena or Evl .
  • test compounds may be assessed for their ability to increase or decrease Mena activity or expression relative to a control assay in which the test compound is absent. Screening assays of the present invention exploit the binding of Mena and Evl to different substrates.
  • test compounds are screened for the ability to compete with Mena binding partners such as ActA, profilin, Zyxin or Vinculin.
  • test compounds are screened for the ability to modulate Mena or Evl activity by increasing or decreasing Mena or Evl expression.
  • labeled Mena or Evl proteins, peptides, or anti-Mena or anti-Evl antibodies are employed to detect expression, localization and/or activity of Mena or Evl associated with normal and/or abnormal cytoskeletal structures or processes, or in association with specific molecular factors involved in cytoskeletal regulation.
  • Mena or Evl expression or activity is detected and/or quantified in a normal cell population or tissue, and these results are compared to Mena or Evl expression or activity detected and/or quantified in a test cell population or tissue (for example a population of cancerous cells or cells from a site of neural injury) .
  • Detection and/or quantification of Mena or Evl expression, localization or activity can be accomplished by a variety of methods, such as by in situ hybridization using anti-Mena or anti-EVl antibodies on embryos or tissue sections or within antibody microinjected cells, by Western blotting or immunoprecipitation using anti-Mena or anti-Evl antibodies in cell or tissue lysates, or by affinity purification using anti-Mena or anti-EVl antibodies bound to a solid phase, among other methods. Comparable methods are disclosed herein, or are elsewhere disclosed and known in the art, for using non-antibody agents to detect and/or quantify Mena or Evl expression or activity.
  • Suitable non-antibody probes for use within these methods include for example oligonucleotide probes that hybridize to Mena or Evl transcripts, labeled binding partners of Mena or Evl such as ActA, zyxin, vinculin or profilin, and synthetic or recombinant peptide analogs of Mena or Evl binding partners, among other useful probe types.
  • Mena and Evl cDNA and oligonucleotide probes may be useful in Northern, Southern, and dot-blot assays for identifying and quantifying the level of expression of Mena or Evl in a cell.
  • Measuring the level of Mena or Evl expression may provide prognostic markers for assessing the growth rate and invasiveness of tumors.
  • birth defects and abortions may result from the absence or expression of an abnormal Mena protein.
  • Mena may be useful in prenatal screening of mothers and/or for in utero testing of fetuses.
  • Differences that are detected and/or quantified between Mena or Evl expression or activity between normal and test cell populations or tissues may be diagnostic of particular disease states or other conditions characterized by aberrant cytoskeletal structure or regulation.
  • cancerous or precancerous test cells such as CML cells
  • an increase of Mena expression compared to control cells is predictive of an increased risk of metastatic disease due to Mena-mediated cell motility and reduction of contact inhibition.
  • the level of Mena expression or activity compared to control cells is predictive of the extent of neural regeneration that can be expected in a particular case, and may also be useful for determining preferred courses of treatment.
  • the presence and pattern of Mena expression in association with the pathogen provides a positive diagnosis of the type of infection, and the level of Mena expression or activity compared to control cells is diagnostic of the severity of infection.
  • Additional diagnostic methods of the invention rely on labeled polynucleotide probes to map the chromosomal locations of Mena, Evl and related genes, to determine linkage of these genes relative to other genes, and to identify genetic defects in these genes among cell populations or individuals. To facilitate these methods, Mena was demonstrated by genetic linkage analysis to be located in the mouse to chromosome la offset 73 in the Jackson backcross hybrid mapping panel.
  • c-Src may be required for proper neurite outgrowth on the cell adhesion molecule LI (Ignelzi et al . , Neuron 12:873-84, 1994).
  • Mammalian c-Abl is a another logical candidate Mena kinase. From studies in NIH3T3 cells, c-Abl is widely perceived as a largely nuclear tyrosine kinase. However, in P19 neurons, Abl is found exclusively in the cytoplasm and in growth cones.
  • Mena contains serine residues aligning to known cyclic nucleotide kinase-dependent phosphorylation sites in VASP (Butt et al . , J. Biol . Chem. 269:14509-17, 1994). These serine residues in Mena flank the central proline-rich region of the protein. Evl contains the amino- terminal site only, while Ena lacks both sites.
  • Screening methods that are particularly useful within the invention include high throughput screening assays designed to identify modulators of Mena or Evl expression or activity.
  • labeled Mena or Evl proteins, peptides, or anti-Mena or anti- Evl antibodies are employed in a similar manner as described above to detect and/or quantify expression or activity of Mena or Evl in comparable test and control samples.
  • Useful control samples in this context generally include a variety of in vivo or in vi tro assay mixtures suitable for detecting and/or quantifying Mena or Evl binding to a selected binding partner, for example ActA, zyxin, vinculin or profilin.
  • Other suitable control samples include in vivo or in vivo assays mixtures suitable for detecting other activities of Mena or Evl , for example subcellular localization of Mena to the surface of a cytopathogenic bacteria or a site of endogenously controlled microfilament formation, or Mena or Evl phosphorylation.
  • Useful test samples within these screening methods contain an added test substance, i.e.
  • the test sample contains suitable amounts of Mena or Evl protein and a selected binding partner under conditions that permit the formation of Mena- or Evl-binding partner complexes in the absence of the test substance.
  • the complexes are then detected and/or quantified according the methods disclosed herein, and these results are compared to the results of detection and/or quantification of Mena- or E l-binding partner complexes formed in the control sample.
  • kits and multicontainer units comprising reagents and components for practicing the assay methods of the present invention.
  • Kits of the present invention may, in addition to reagents for detecting Mena and Evl , contain enzymatic reagents such as reverse transcriptase or polymerase; suitable buffers; nucleoside triphosphates; suitable labels for labeling the reagents for detecting Mena and Evl and developing reagents for detecting the signal from the label.
  • kits of the present invention contain sequence-specific oligonucleotide primers for detecting polynucleotide molecules encoding Mena and Evl .
  • kits containing Mena and Evl sequence- specific probes contain antibodies useful for detecting Mena and Evl (or mutants thereof) in a sample.
  • kits contain Mena- and/or Evl-specific antibodies for detecting Mena and Evl protein.
  • the Mena and E l-specific antibodies may be labeled or may be detected by binding to a secondary antibody.
  • the antibody reagents may be provided in separate container or may be provided in combination in a series of containers.
  • the kits may also contain instructions for carrying out the assay and/or additional containers suitable for carrying out the reactions of the assay.
  • the complex structure of the Mena and Evl proteins, and the various biological activities of EVHl and other functional domains of Mena or Evl provide for a broad array of potential screening assays and assay formats.
  • ligand binding assays using immobilized Mena or Evl proteins or fusion proteins are preferred for high throughput screening purposes.
  • labeled Mena or Evl proteins or Mena or Evl based peptides or fusion proteins are immobilized on a solid phase, for example, an N-Mena-Gst fusion protein immobilized on a glutathione-agarose bead matrix such as glutathione SEPHAROSE (Pharmacia Biotech, Inc., Piscataway, NJ) .
  • the control assay sample is completed by exposing the bound N-Mena-Gst fusion protein to a N-Mena ligand, for example ActA, zyxin or vinculin.
  • the ligand may be provided in a purified form, or as a component of a ligand- containing mixture, for example a cell or tissue lysate.
  • N-Mena-ligand complexes are detected and/or quantified following exposure of the ligand to the bound fusion protein using any of a variety of detection or quantification methods disclosed herein, for example by a selected immunoassay using antibodies to ActA, zyxin or vinculin.
  • the test assay sample is prepared in a comparable fashion, by exposing the bound N-Mena-Gst fusion protein to an N-Mena ligand under conditions that permit formation of N-Mena- ligand complexes in the control assay sample.
  • test substances prior to detection and/or quantification of N-Mena- ligand complexes in the control assay sample, a test substance is included therein.
  • the test substances is added in the form of a purified agent, however it is also contemplated that test substances useful within the invention may include substances present throughout the handling of test sample components, for example host cell factors that are present in a cell lysate used for generating the test sample.
  • host cell factors may be segregated between the test and control samples for example by using different cell types for preparing lysates, where the cell type used for preparing the test sample expresses a putative test substance that is not expressed by the cell type used in preparing the control sample.
  • a particularly useful set of test and control cell types in this context are P19 embryonic carcinomal cells that have and have not been induced to undergo neural differentiation by retinoic acid.
  • test and control samples include reagents that are microinjected into cells to yield an in vivo assay mixture.
  • Gst-N-Mena is microinjected into PtK 2 cells seeded to semi- confluency on Cellocate coverslips (Eppendorf, Hamburg, Germany) .
  • Labeled anti-Gst or anti-N-Mena antibodies are also injected or otherwise delivered (e.g. by microporation or liposomal transfection) into the cells.
  • a control sample of cells thus treated the pattern and/or intensity of antibody labeling is detected.
  • a test substance such as the ActA sequence SEPSSFEFPPPPTDEELRLA (SEQ ID NO. 12) , is delivered into the cell prior to detecting the pattern and/or intensity of antibody labeling.
  • results of this form of assay can be readily determined based on simple qualitative observations, for example by immunofluorescence visualization of Gst-N-Mena depletion from focal contacts, or from the surface of Listeria or other pathogens in infected cells, confined to the test sample.
  • flexneri a gene encoding an outer membrane protein variously denoted VirG or IcsA has been identified and is required for host actin recruitment to form actin halos and comets to spread from cell to cell (Pollard, Current Biology 5:837-40, 1995, which is incorporated herein by reference in its entirety) .
  • pathogens which appear to share closely related actin recruitment mechanisms, there are a variety of different pathogens having apparently distinct mechanisms and/or used for host cell actin recruitment.
  • EPEC lethal enteropathogenic E. coli
  • enterohemorrhagic form of E E.
  • EHEC EHEC
  • EHEC EHEC
  • EHEC EHEC
  • host cell tyrosine kinases and actin accumulation leading to profound host cytoskeletal disruption (reviewed by Donnenburg et al . , J. Clin . Invest . £32:1418-24 , 1995, which is herein incorporated by reference in its entirety)
  • diverse viral pathogens have recently been discovered to also cause profound changes in cytoskeletal dynamic processes.
  • the HIV virus spreads between cells via syncitia formation which involves both cell motility and reorganization of the cytoskeleton accompanied by condensation of F-Actin (Sylwester et al . , J. Cell . Sci .
  • vaccinia virus in its intracellular enveloped form induces the formation of actin tails that are "strikingly similar" to those seen in Listeria, Shigella and Rickettsia infections (Cudmore et al . , Nature
  • the screening methods of the invention will be particularly useful to identify negative modulators of Mena or Evl expression and/or activity. Once identified, these Mena or Evl inhibitors will supplement existing tools and methods within the invention for preventing and treating such pathogenic diseases.
  • Mena or Evl based recombinant polynucleotide expression constructs which include a polynucleotide insert encoding a Mena or Evl protein or peptides, fusion protein or the like operably incorporated within an expression vector capable of directing expression of the polynucleotide insert in a mammalian host cell.
  • Such constructs may include native or mutagenized forms of the Mena or Evl protein or of selected polypeptide portions of Mena or Evl such as the EVHl functional domain.
  • Various such constructs are provided, including constructs that incorporate polynucleotide sequences encoding the native 140 kD, 60 kD, 80 kD or 88 kD Mena isoforms, Evl or consensus sequences derived from SEQ ID NO: 9.
  • Cells transformed with these constructs may contain for example altered Mena coding sequences that result in the expression of a Mena protein that is not capable of binding to one or more of the EVHl domain ligands of Mena, including ActA, zyxin and vinculin, or which has enhanced binding to profilin that results in a phenotype characterized by hyperfunctional actin polymerization.
  • the subject cell lines and animals find uses in screening for candidate therapeutic agents capable of either substituting for a function performed by Mena or correcting the cellular defect caused by a defective Mena .
  • polynucleotide molecules of the present invention may be joined to reporter genes, such as -galactosidase or luciferase, and inserted into the genome of a suitable embryonic host cell such as an mouse embryonic stem cell by, for example, homologous recombination (for review, see Capecchi, Trends in Genetics 5 : 70-76, 1989; which is incorporated by reference). Cells and cell lines expressing the subject molecules may then be obtained and used, for example, for screening for compounds that increase or decrease expression of the reporter gene.
  • reporter genes such as -galactosidase or luciferase
  • mice are generated by replacing the murine Mena or Evl coding region with the -galactosidase reporter gene and the neomycin resistance gene to assess the consequences of eliminating the murine Mena or Evl protein, and to examine the tissue distribution of Mena or Evl in fetal and post-natal mice.
  • These "Knock out" mice are useful for example as model systems for screening compounds that may developmentally, spatially and/or quantitatively alter the expression of the reporter gene. Such mice may be used to study methods to rescue homozygous mutants and as hosts to test transplant tissue for treating diseases or other conditions characterized by aberrant regulation of cytoskeletal dynamic processes.
  • the invention provides a range of therapeutic methods for preventing and/or treating diseases and other conditions that involve aberrant cytoskeletal regulation, for example pathogenic infection, traumatic neural injury and cancer.
  • Therapeutic methods of the invention variously utilize Mena or Evl encoding polynucleotides, Mena or Evl proteins and peptides, Mena or Evl binding antibodies, Mena or Evl based expression constructs, mammalian host cells transformed to express Mena or Evl insert sequences, and other therapeutic agents generally characterized as modulators of Mena or Evl expression or activity, including triplex forming oligonucleotides, antisense polynucleotides and ribozymes that specifically target Mena and/or Evl polynucleotides.
  • agents that inhibit Mena or Evl expression or activity In preferred therapeutic methods directed to the treatment of pathogenic infection and cancer, it is useful to employ agents that inhibit Mena or Evl expression or activity and which thereby can eliminate or impair aberrant recruitment of Mena by pathogens, or interrupt Mena expression or activity associated with cancerous cell migration.
  • agents that induce or enhance Mena or Evl expression or activity In preferred therapeutic methods directed to the treatment of traumatic neural injury and other injury conditions where it is desirable to promote cell migration for neural regeneration or wound healing, it is useful to employ agents that induce or enhance Mena or Evl expression or activity.
  • Useful agents in this context generally include agents that enhance Mena or Evl binding to its partners or otherwise enhance Mena or Evl expression or activity, for example mutant Mena or Evl based expression constructs that induce or enhance Mena or Evl expression or activity in targeted cells.
  • Therapeutic substances which can serve as inhibitors or antagonists of Mena or Evl include, but are not limited to, compounds capable of inhibiting the formation of Mena-profilin or Mena-proline-rich binding partner, compounds that reduce or inhibit the activity of Mena, and compounds that interfere with the expression of Mena protein.
  • Such agents may include chemical compound inhibitors of Mena, protein or peptide Mena antagonists, and molecules that inhibit the expression of Mena such as triplex forming oligonucleotides, antisense polynucleotides, ribozymes, etc.
  • antisense polynucleotides are described generally in, for example, Mol and Van der Krul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, NY, 1992, which is incorporated by reference herein in its entirety.
  • Suitable antisense oligonucleotides are at least 11 nucleotides in length and up to and including the upstream untranslated and associated coding sequences of Mena .
  • the optimal length of antisense oligonucleotides is dependent on the strength of the interaction between the antisense oligonucleotides and their complementary sequence on the mRNA, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, the presence of secondary and tertiary structure in the mRNA and/or in the antisense oligonucleotide and the preferred delivery mode.
  • soluble antisense oligonucleotides have been used to inhibit transcription/translation of a target gene (Ching et al . , Proc. Natl . Acad . Sci . U. S . A .
  • Suitable target sequences for antisense polynucleotides include intron-exon junctions (to prevent proper splicing) , regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression.
  • a particularly preferred target region for antisense polynucleotides is the 5 ' untranslated region of the gene of interest, for example the Mena gene.
  • Antisense polynucleotides targeted to the Mena gene may also be prepared by inserting a DNA molecule containing the target DNA sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself.
  • the expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense polynucleotides.
  • antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by a variety of means including electroporation (e.g., as described in Yang et al . , Nucl . Acids . Res .
  • Stabilizing agents include intercalating agents that are covalently human EST accession #T80305 (obtained from , Washington University-Merck EST Project IMAGE Consortium, LLNL). The EST was used to screen the library as described above. A murine cDNA was isolated and sequenced as previously described in detail. The cDNA (nucleotide sequence and deduced amino acid sequence in SEQ ID NO:7 and SEQ ID NO: 8) was predicted to encode a 393 amino acid protein. Based on sequence analysis the cDNA was termed Ena- VASP Like (Evl).
  • Evl Ena- VASP Like
  • Evl contains the amino-terminal site only, while Ena lacks both sites.
  • the sequence GPPPPP (SEQ ID NO: 13), which mediates binding of VASP to the actin- monomer sequestering protein profilin (Reinhard et al., EMBO J. 14: 1583-1589, 1995) is present twice in Mena and once in Evl.
  • the carboxy terminal homology domain (EVH2, spanning amino acids 507-733 in the consensus line), contains a putative G-actin binding sequence, and a conserved charge cluster.
  • Mena also contains a striking five amino-acid repeat region with the consensus LERER (SEQ ID NO: 10), located between the EVHl domain and the first conserved serine phosphorylation site at amino acid 236 of SEQ ID NO: 2.
  • the extended helical structure predicted for this repeat may function as a protein-binding interface, or serve to separate the EVHl domain from the proline-rich core of Mena.
  • the Mena cDNA was used to probe the Jackson backcross hybrid mapping panel
  • Mena The Jacson Laboratory, Bar Harbor, ME
  • Mena was localized to the mouse chromosome la offset 73 which is syntenic to a human chromosomal position 1Q21-23 (Stanier et al., Genomics 26: 473-478, 1995).
  • Mena-specific antibodies were developed. DNA fragments encoding amino acids 6-170 of SEQ ID NO: 2 (N-Mena) or amino acids 440- 537 of SEQ ID NO: 2 (C-Mena) were amplified from the Mena cDNA using polymerase chain reaction amplification. The fragments were each cloned into pGex2T (Smith and Johnson Gene 67: 31-40, 1988, which is incorporated herein by reference in its entirety) and transformed into a bacterial host to permit the expression of Mena-glutathione S- transferase fusion proteins.
  • pGex2T Smith and Johnson Gene 67: 31-40, 1988, which is incorporated herein by reference in its entirety
  • the fusion proteins were purified on a glutathione-coupled beaded agarose matrix (Glutathione SEPHAROSE, Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions. Rabbits were immunized and boosted with the Mena fusion proteins, and sera collected as described by Harlow and Lane in Antibodies A Laboratory Manual (Cold Spring Harbor, NY, Cold Spring Harbor Press 1988), which is incorporated herein by reference in its entirety. Affinity purified antisera raised against the peptide LKEELIDAIRQELSKSNTA (SEQ ID NO: 1 1) were produced by Quality Controlled Biochemicals, Inc. (Hopkins, MA). Purified platelet VASP protein was not recognized by any of the Mena antibodies tested.
  • Glutathione SEPHAROSE Pharmacia Biotech, Inc., Piscataway, NJ
  • Example 3 Distribution of Mena The distribution of Mena protein isoforms during a portion of mouse embryogenesis was determined by Western blot analyses of protein from dissected heads or bodies of embryo-lysates taken from stages El 0 to El 5 or from lysates of whole E9 embryos. Protein extracts were from cells or dissected embryos were prepared in ice cold RIPA buffer (25 mM Tris (pH 7.5), 150 mM NaCI, 1 % NP40, 0.5% Deoxycholate, 0.1% SDS) + 1 mM PMSF, Aprotinin and ImM NaVO . Proteins were quantified using the Pierce protein assay kit (Pierce Chemical Company, Rockford, IL).
  • Immunoprecipitations were performed with 1ml of 1 mg/ml of head lysate using 5 ⁇ l of Anti-N-Mena (from rabbit 2188) or 2188 pre-immune sera. Fifty micrograms of protein were used for Western blotting of total cell lysates. Gel electrophoreses and Western blotting on polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA) were performed as described by Harlow and Lane (ibid.) and according to the manufacturer's instructions. Signals were visualized using chemiluminescence (Dupont Co. (NEN Life Science Products), Wilmington, DE).
  • the 140 kD band was also enriched during the course of retinoic acid-induced differentiation of the P19 embryonic carcinomal stem cells into neurons (Rudnicki and McBurney, Cell Culture Methods and Induction of Differentiation of Embryonal Carcinoma Cell Lines. In Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. Robertson, ed., Oxford, England, pp. 19-47, 1987; which is incorporated herein by reference in its entirety).
  • Anti-Mena Western blot analyses of lysates from P19 embryonic carcinomal cells at 0,3,5, and 7 days after treatment with retinoic acid demonstrated that the 140 kD form of Mena was induced during the differentiation process.
  • the translation products were subjected to gel electrophoresis, and the gels were fixed and treated with Amplify (Amersham, Arlington Heights, IL). The fixed gels were dried and quantified using a phosphoimager (Molecular Dynamics, Sunnyvale, CA). Translation of the Mena cDNA gave rise to a protein that co- 4U
  • the 80 kD signal is a broadly expressed form of Mena
  • the 140 kD signal is a Mena isoform enriched in, or specific to neural cell types and produced by alternative splicing.
  • the 88 kD signal is immunoreactive with all anti-Mena antibodies tested, and may represents another splice variant, or a post-translationally modified form of Mena.
  • Neural Mena 4 contained an exon that introduces 244 amino acids (corresponding to amino acid 239 through amino acid 482 of SEQ ID NO: 4) between amino acids 238 and 239 of (SEQ ID NO: 2).
  • the nucleotide sequence and deduced amino acid sequences of neural Mena are shown in (SEQ ID NO: 3 and SEQ ID NO:4).
  • Two other isoforms, neural Mena ++ and neural Mena +++ contained the exon insertion described for Neural Mena + and either a exon insertion between amino acids 1 16 and 1 17 of SEQ ID NO: 2.
  • the deduced amino acid sequences of neural Mena ++ and neural Mena +++ are shown in SEQ ID NO: 5 and SEQ ID NO:6, respectively.
  • Mena expression was examined in the rat embryo line Rat2 (ATCC CRL 1764). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies (GIBCO/BRL), Gaithersburg, MD) supplemented with 10% FCS. In Rat2 cells, Mena migrates as an 80 kD doublet. Treatment of Mena protein with purified protein kinase A (New England Biolabs, Beverly, MA) according the manufacturer's instructions caused a quantitative conversion of the 80 kD form of Mena to the slower migrating form of the doublet.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Rat2 cells Mena migrates as an 80 kD doublet. Treatment of Mena protein with purified protein kinase A (New England Biolabs, Beverly, MA) according the manufacturer's instructions caused a quantitative conversion of the 80 kD form of Mena to the slower migrating form of the doublet.
  • the Mena cDNA was translated in vitro and labeled with 35 S-methionine as described previously.
  • the translation product migrated as an 80 kD doublet.
  • Addition of protein kinase A shifted the mobility of the lower band to that of the upper, indicating that this doublet was caused by partial serine phosphorylation in the lysate.
  • Rat2 cells were grown in DMEM (Life Technologies (GIBCO BRL), Gaithersburg, MD) supplemented with 10% FCS on glass coverslips coated with 5 ⁇ g/cm 2 fibronectin (Sigma Biosciences, St. Louis, MO). Cells were fixed with either 3% or ice-cold 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes. The fixed cells were permeabilized with 0.2% (v/v) Triton X-100 in PBS and stained as described (Harlow and Lane, ibid.). Mena-specific antisera was used at dilution of 1 :400.
  • FITC Fluorescein
  • Texas Red secondary antibody conjugates were purchased from Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and used at a dilution of 1 : 100.
  • Phosphotyrosine and neurofilaments detected using mab4G10 were purchased from Jackson Immunoresearch Laboratories, Inc., West Grove, PA.
  • Bodipy phalloidin (Molecular Probes Inc., uptake of the oligonucleotides by cells.
  • lipids include cationic lipids used for lipofection such as N-[l-(2,3- dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) .
  • DOTMA N-[l-(2,3- dioleyloxy)propyl-N,N,N-trimethylammonium chloride
  • DOPE dioleoyl phosphatidylethanolamine
  • One skilled in the art may determine the particular lipid formulation or concentration that will be effective for enhancing the uptake of the oligonucleotide.
  • Mena or Evl inhibitors or agonists may be used in combination with other compounds that are useful, for example in chemotherapy or as antibiotics.
  • These compounds include standard chemotherapeutic agents such as platin compounds (e.g. cisplatin) and antibiotics such as penicillin, tetracycline or antiviral agents such as protease inhibitors and the like.
  • Mena or Evl based polynucleotide expression constructs disclosed herein are employed in methods for modulating the expression of Mena or Evl , disrupting Mena or Evl expression or inducing ectopic expression or Mena or Evl genes and related polynucleotide sequences in the context of gene therapeutic methods involving mammalian host cells.
  • Mena is ectopically expressed or over expressed in mammalian cells to induce cytoskeletal changes, including for example formation of dense F-actin clusters, cell surface protrusions characterized by Mena-actin core structures, and filopodial extension and cytoplasmic bridge formation between neighboring cells.
  • a suitable gene therapy vector delivery system e.g., a retroviral vector
  • a microinjection technique see, for example, Tarn, Basic Life Sciences 37:187-194, 1986, which is incorporated by reference herein in its entirety
  • a transfection method e.g., naked or liposome encapsulated DNA or RNA
  • Gene transfer vectors e.g., retroviral vectors, and the like
  • Gene therapy may be used to correct traumatic neural injury that has resulted in loss of motor or sensory neural function.
  • the introduction method may be chosen to achieve a transient expression of Mena in the host cell, or it may be preferable to achieve constitutive, tissue specific, or inducible expression.
  • Methods of treatment employing transformed host cells of the present invention are useful in a variety of in vivo settings, for example, for transplantation at sites of traumatic neural injury where motor or sensory neural activity has been lost.
  • the polynucleotide constructs and insert sequences of the invention provide the possibility of specific gene therapy for the treatment of certain neurological disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, in which a population of neurons have been damaged.
  • Representative patient populations that may benefit from transplantation include, but are not limited to, patients with hearing or vision loss due to optical or auditory nerve damage, patients with peripheral nerve damage and loss or motor or sensory neural function, individuals with diseases such as cancer or developmental defects relating to aberrant cytoskeletal dynamic function, and patients with brain or spinal cord damage from traumatic injury.
  • donor cells fora patient having CML are obtained from a normal stem cell population in the same patient or in a donor patient and are then transformed or transduced with a mutant Mena or Evl nucleotide sequence.
  • a preferred Mena mutant in this context can be designed to express a hyperfunctional EVHl domain that has a strongly enhanced binding affinity for ligands such as zyxin or vinculin that mediate Mena function in cytoskeletal dynamic processes.
  • a second mutation can be introduced in a separate functional domain, for example the proline rich, profilin binding domain, that renders the double mutant protein incapable of binding profilin to promote actin polymerization, while it remains tightly bound to its EVHl domain ligands and thereby prevents or impairs binding and function of endogenous Mena in the donor cells.
  • the transformed cells that are rendered resistant to the motility defects and loss of contact inhibition of CML cells are then returned to the patient, for example in the manner routinely performed during bone marrow transplants.
  • gene transfer may be achieved by introducing the sequences of the present invention directly to the site of the traumatic injury.
  • Mena and Evl A cDNA encoding Mena was identified by screening a cDNA library made from mouse embryonic stem cells (Chen et al . , Proc . Natl . Acad. Sci . USA 92:7819-7823, 1994). Filter lifts of the library were prepared on nylon membranes (HYBOND, Amersham, Arlington Heights, IL) . Library screening, subcloning and polymerase chain reactions were performed, and standard solutions prepared essentially as described by Sambrook et al . (Molecular Clonings : A Laboratory Manual , Cold Spring Harbor, NY, 1989, which is incorporated herein by reference in its entirety) .
  • the library was screened with a 32 P- labeled (Amersham, Arlington Heights, IL) Eco Rl/Sph I fragment containing the first 106 codons of Ena (Gertler et al . , Genes Dev. 9:521-533, 1995).
  • the filters were hybridized in a buffer containing: 1.5 x SSPE, 7% SDS, 10% PEG8000 and lxlO 6 cpm/ml of the 32 p-labeled Ena probe overnight at 42°C.
  • the filters were washed repeatedly at 42°C in a buffer containing 2 x SSC, 1% SDS followed by washes at 60°C in a buffer containing 0.5 x SSC, 1% SDS.
  • cDNA inserts were subcloned into pBSKSII (Stratagene Cloning Systems, La Jolla, CA) .
  • the inserts were prepared and sequenced on both strands using an Applied Biosystems DNA Sequencer model 373A (Applied Biosystems, Foster City, CA) according to the manufacturer's guidelines. Analysis of overlapping cDNAs indicated that these sequences contained an open reading frame with a predicted amino acid sequence of 541 amino acids (SEQ ID NO. I and SEQ ID NO. 2) .
  • the complete cDNA was termed Mammalian Ena (Mena) . Sequence analysis was carried out using the Genetics Computer Group software package (Devereaux et al . , Nuc . Acids Res . 12 : 387-395, 1994) . The analysis showed significant similarity to Drosophila Ena on the amino acid level.
  • Initial sequence alignments were generated using the PILEUP program and modified using the LINEUP sequence editor
  • the Mena sequence was used to search the GenBank databases at the National Center for Biotechnology Information (Bethesda, MD) using the BLAST program. Two related sequences were identified: VASP (Harmer et al . , EMBO J. 14 : 19-21 . 1995), and human EST accession #T80305 (obtained from, Washington University-Merck EST Project IMAGE Consortium, LLNL) . The EST was used to screen the library as described above. A murine cDNA was isolated and sequenced as previously described in detail. The cDNA (nucleotide sequence and deduced amino acid sequence in SEQ ID NO: 7 and SEQ ID NO. 8) was predicted to encode a 393 amino acid protein.
  • Ena-VASP Like (Evl ) . Alignment of Mena , Evl , human VASP, and Ena revealed two distinct blocks of similarity flanking a proline-rich core. The consensus sequence is shown in SEQ ID NO: 9. The greatest degree of amino acid identity is in amino terminal 113 amino acid of SEQ ID NO: 9, which is termed "Ena-VASP homology domain 1" (EVHl) . A pair-wise comparison among the 5 sequences throughout the EVHl domain revealed that Mena and Ena are the most similar of the four Ena-VASP family members.
  • Mena also contains a striking five amino-acid repeat region with the consensus LERER (SEQ ID NO: 10) , located between the EVHl domain and the first conserved serine phosphorylation site at amino acid 236 of SEQ ID NO. 2.
  • the extended helical structure predicted for this repeat may function as a protein-binding interface, or serve to separate the EVHl domain from the proline-rich core of Mena .
  • Both the EVH2 and the LERER SEQ ID NO.
  • Mena cDNA was used to probe the Jackson backcross hybrid mapping panel (The Jackson Laboratory, Bar Harbor, ME) to determine the chromosomal location of the Mena gene. Mena was localized to the mouse chromosome la offset 73 which is syntenic to a human chromosomal position 1Q21-23 (Stanier et al . , Genomics 26:473-478, 1995).
  • Mena-specific antibodies were developed. DNA fragments encoding amino acids 6-170 of SEQ ID NO. 2 (N-Mena) or amino acids 440-537 of SEQ ID NO. 2 (C-Mena) were amplified from the Mena cDNA using polymerase chain reaction amplification. The fragments were each cloned into pGex2T (Smith and Johnson Gene 67:31-40, 1988, which is incorporated herein by reference in its entirety) and transformed into a bacterial host to permit the expression of Mena-glutathione Transferase fusion proteins.
  • pGex2T Smith and Johnson Gene 67:31-40, 1988, which is incorporated herein by reference in its entirety
  • the fusion proteins were purified on a glutathione-coupled beaded agarose matrix (Glutathione SEPHAROSE, Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions. Rabbits were immunized and boosted with the Mena fusion proteins, and sera collected as described by Harlow and Lane in Antibodies A Laboratory Manual (Cold Spring Harbor, NY, Cold Spring Harbor Press 1988) , which is incorporated herein by reference in its entirety. Affinity purified antisera raised against the peptide LKEELIDAIRQELSKSNTA (SEQ ID NO: 11) were produced by Quality Controlled Biochemicals, Inc. (Hopkins, MA) . Purified platelet VASP protein was not recognized by any of the Mena antibodies tested.
  • Glutathione SEPHAROSE Pharmacia Biotech, Inc., Piscataway, NJ
  • Mena protein isoforms during a portion of mouse embryogenesis was determined by Western blot analyses of protein from dissected heads or bodies of embryo-lysates taken from stages ElO to E15 or from lysates of whole E9 embryos. Protein extracts were from cells or dissected embryos were prepared in ice cold RIPA buffer (25 mM Tris (pH 7.5), 150 mM NaCI, 1% NP40, 0.5% Deoxycholate, 0.1% SDS) + 1 mM PMSF, Aprotinin and 1 mM NaV0 4 . Proteins were quantified using the Pierce protein assay kit (Pierce Chemical Company, Rockford, IL) .
  • Immunoprecipitations were performed with 1 ml of 1 mg/ml of head lysate using 5 ⁇ l of Anti-N-Mena (from rabbit 2188) or 2188 pre-immune sera. Fifty micrograms of protein were used for Western blotting of total cell lysates. Gel electrophoreses and Western blotting on polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA) were performed as described by Harlow and Lane (ibid.) and according to the manufacturer's instructions. Signals were visualized using chemiluminescence (Dupont Co. (NEN Life Science Products) , Wilmington, DE) .
  • the 140 kD band was also enriched during the course of retinoic acid- induced differentiation of the P19 embryonic carcinomal stem cells into neurons (Rudnicki and McBurney, Cell Cul ture Methods and Induction of Differentiation of Embryonal Carcinoma Cell Lines . In Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, E. Robertson, ed., Oxford, England, pp. 19-47, 1987; which is incorporated herein by reference in its entirety) .
  • Anti-Mena Western blot analyses of lysates from P19 embryonic carcinomal cells at 0, 3, 5, and 7 days after treatment with retinoic acid demonstrated that the 140 kD form of Mena was induced during the differentiation process.
  • the mobility of the 80 kD and 140 kD Mena isoforms was slower than that predicted by their sequence (60 kD and 83 kD respectively) , perhaps due to structural effects of their large proline content.
  • the Mena cDNA was translated in vi tro to produce 35 S- labeled Mena protein using the TNT coupled transcription/translation system (Promega Corp, Madison, CA) .
  • the translation products were subjected to gel electrophoresis, and the gels were fixed and treated with Amplify (Amersham, Arlington Heights, IL) .
  • the fixed gels were dried and quantified using a phosphoimager (Molecular Dynamics, Sunnyvale, CA) .
  • Mena cDNA Translation of the Mena cDNA gave rise to a protein that co-migrates with the 80 kD band, while expression of the neural Mena cDNA (Example 4) in fibroblasts produced a signal at 140 kD in addition to the endogenous doublet at 80 kD.
  • the 80 kD signal is a broadly expressed form of Mena
  • the 140 kD signal is a Mena isoform enriched in, or specific to neural cell types and produced by alternative splicing.
  • the 88 kD signal is immunoreactive with all anti-Mena antibodies tested, and may represent another splice variant, or a post- translationally modified form of Mena.
  • Neural Mena + contained an exon that introduces 244 amino acids (corresponding to amino acid 239 through amino acid 482 of SEQ ID NO. 4) between amino acids 238 and 239 of (SEQ ID NO. 2) .
  • the nucleotide sequence and deduced amino acid sequences of neural Mena are shown in (SEQ ID NO. 3 and SEQ ID NO . 4) .
  • neural Mena ++ and neural Mena +++ contained the exon insertion described for Neural Mena+ and either a exon insertion between amino acids 116 and 117 of SEQ ID NO. 2.
  • the deduced amino acid sequences of neural Mena ++ and neural Mena +++ are shown in SEQ ID NO. 5 and SEQ ID NO.6, respectively.
  • Mena Phosphorylation of Mena
  • the phosphorylation status of Mena was determined by immunoprecipitation.
  • RIPA lysates from E12 embryonic heads were immunoprecipitated using anti-N terminal Mena antibodies or pre-immune sera. The precipitates were analyzed by Western blotting as described above with the 4G10 anti-phosphotyrosine monoclonal antibody.
  • Mena immunoprecipitates from E12 head lysates contained an anti-phosphotyrosine reactive signal that co-migrates with the 140 kD form.
  • the 80 and 88 kD forms contained no detectable phosphotyrosine .
  • Mena expression was examined in the rat embryo line Rat2 (ATCC CRL 1764). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Life Technologies (GIBCO/BRL), Gaithersburg, MD) supplemented with 10% FCS. In Rat2 cells, Mena migrates as an 80 kD doublet. Treatment of Mena protein with purified protein kinase A (New England Biolabs, Beverly, MA) according to the manufacturer's instructions caused a quantitative conversion of the 80 kD form of Mena to the slower migrating form of the doublet.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the Mena cDNA was translated in vi tro and labeled with 35 S-methionine as described previously.
  • the translation product migrated as an 80 kD doublet.
  • Addition of protein kinase A shifted the mobility of the lower band to that of the upper, indicating that this doublet was caused by partial serine phosphorylation in the lysate.
  • Example 6 Subcellular Localization of Mena The subcellular distribution of Mena in non-neural cell types was determined by immunofluorescence. Rat2 cells were grown in DMEM (Life Technologies (GIBCO/BRL) , Gaithersburg, MD) supplemented with 10% FCS on glass coverslips coated with 5 ⁇ g/cm 2 fibronectin (Sigma Biosciences, St. Louis, MO). Cells were fixed with either 3% or ice-cold 4% paraformaldehyde in phosphate buffered saline
  • PBS Phosphotyrosine and neurofilaments detected using mab4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), and mab 2H3D12 (obtained from Tom Jessell, Columbia University, New York, NY).
  • Bodipy phalloidin (Molecular Probes Inc., Eugene, OR) was used to stain F-actin, DAPI (Sigma, St. Louis, MO) was used to stain nuclei, and Vectashield mounting media (Vector Labs, Burlingame, CA) were used to in the preparation of the images. Three dimensional microscopic images were captured and processed using a Deltavision microscope and software (Applied Precision Inc., Mercer Island, WA) .
  • Phosphotyrosine appeared distal to Mena, followed by a region of overlap, and a proximal region that contains Mena .
  • Mena unlike VASP, high levels of Mena are not observed along the length of actin stress fibers.
  • the staining was specific for Mena, as signal was not observed in cells stained with preimmune sera, or in macrophages which contain VASP, but not Mena .
  • Similar Mena staining was observed with both anti-N-Mena, and anti-C-Mena antibodies.
  • the distribution of Mena was compared to phosphotyrosine, a marker for focal adhesions (Maher et al . , Proc . Natl . Acad . Sci . USA
  • Mena was restricted to the proximal portions of the phosphotyrosine domain in most focal contacts, at the ends of F-actin stress fibers. Therefore, Mena distribution overlaps with molecules that are receiving extracellular matrix-induced signals and with regions of microfilament assembly.
  • Example 7 EHVl domain binding To investigate whether the highly conserved EVHl domain mediates an interaction with zyxin or vinculin, molecules that may recruit Mena to focal adhesions in fibroblasts or related structures in other cell types) , the EVHl domain was used to purify proteins from embryo cell lysates. N-Mena-Gst, the Gst- fusion protein containing amino acids 6-170 of Mena (described in Example 2) encompasses the EVHl domain.
  • N-Mena-Gst or Gst alone was immobilized on a glutathione-coupled beaded agarose matrix (Glutathione SEPHAROSE, Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's instructions.
  • the N-Mena matrix was mixed with 1 ml of 1 mg/ml of RIPA lysates of E12 heads, or human erythrocytic leukemia cells (HEL) (described above) and incubated overnight at 4°C. Samples were washed three times in RIPA, and bound proteins analyzed by Western blot analysis with antibodies to zyxin (obtained from Dr.
  • N-Mena is capable of binding to zyxin and vinculin.
  • PtK 2 potoroo kidney epithelial cells, (ATCC CCL 56) were seeded in Modified Essential Medium (MEM; Life Technologies (GIBCO/BRL) , Gaithersburg, MD) supplemented with 10% FCS, glutamine and nonessential amino acids to semi confluency onto CELLocate coverslips (Eppendorf, Hamburg, Germany) two days prior to microinjection.
  • MEM Modified Essential Medium
  • FCS FCS
  • glutamine nonessential amino acids to semi confluency onto CELLocate coverslips
  • Purified Gst-N terminal Mena fusion protein was microinjected into the cytoplasm of the cells and GST alone served as a control. After injection, the cells were returned to the incubator for 15 to 30 minutes before fixation.
  • MES- buffer 0.1 M MES, 4 % PEG 6000 (w/v), 1 mM MgCl 2 (pH 7.6)
  • MES-buffer 0.2% Triton X-100
  • cells were fixed with paraformaldehyde, and then incubated 30 minutes each with primary and fluorescently labeled secondary antibodies and phalloidin.
  • Microinjected Gst-N terminal Mena fusion protein was detected using polyclonal anti-GST antibodies.
  • Fluorescence photomicroscopy was carried out on a Zeiss Axiophot with appropriate filter sets for epifluorescence detection of FITC and rhodamine signals.
  • the microinjection of N-Mena-Gst fusion protein into PtK 2 cells demonstrated that the N-Mena sequences directed the fusion protein to focal adhesions.
  • Gst alone was injected, diffuse signal was observed throughout the cell.
  • a peptide corresponding to amino acids 293-312 of the ActA sequence was also injected into PtK 2 cells.
  • the ActA peptide (SEQ ID NO. 12) was first coupled to ovalbumin as described previously (Pistor et al . , Curr. Biol . 5:517-525, 1995; which is incorporated herein by reference in its entirety) , and the conjugate was microinjected along with rhodamine- coupled BSA as a marker protein. The cells were then returned to the incubator for 15 to 30 minutes before fixation.
  • Example 8 Mena Modulates F-actin Dynamics To examine whether increased concentration of Mena protein might stimulate actin nucleation and polymerization, a retroviral vector was used to drive increased levels, or ectopic expression of the different Mena isoforms in Rat2 cells. Rat2 cells were grown under conditions described previously. Retroviruses were constructed by inserting full length Mena cDNAs (Mena, neural Mena, Isoform 1 or Isoform 2) into the pBABE vector (Morgenstern and Land, Nuc . Acids Res . 18:3587-3596, 1990, which is incorporated herein by reference in its entirety) , packaged and used to infect fibroblasts as described Morgenstern and Land (ibid.).
  • neural Mena was particularly dramatic. As expected, individual cells in the experimental population expressed varying degrees of neural Mena as judged by fluorescence intensity. Similar increases in Mena immunofluorescence intensity across the population were observed with the 80 kD form, or any of the three neural Mena variants. Western analysis indicated that, over the whole population, the cumulative amount of ectopic neural Mena protein produced was roughly equivalent to the endogenous content of the 80 kD Mena isoform. About 25% of cells had detectable levels of Mena immunoreactivity outside focal contacts. These cells often contained concentrated pools of immunoreactivity that overlapped with dense accumulations of F-actin.
  • Projection of a three dimensional rendering of the image at a 45° angle indicated that the Mena-actin structures were protuberances from the cell surface.
  • Scanning electron microscopy confirmed the presence of cell-surface projections on a subset of cells from a similarly infected pool, but not in the corresponding control.
  • An optical section taken at the plane of cell -substratum contact showed a relatively normal distribution of Mena, mainly in focal contacts and at the cell periphery, and F-actin, mainly in typical stress fibers.
  • An optical section taken 3.8 ⁇ M above the substratum contact indicated the Mena- induced projections extend above the top of the nuclei.
  • Mena and F-actin staining at this level indicated that Mena is distributed at the periphery of the F-actin, potentially at the site of F-actin polymerization and nucleation in these projections. These data indicate that, when ectopically expressed in fibroblasts, the neural form of Mena can direct the formation of cell surface projections.
  • Example 9 Mena Interacts with Profilin and SH3 The ability of Mena to interact with SH3 and profilin binding was determined by using assays were described by Reinhard et al . (ibid., 1995) and Weng et al . (J. Biol .
  • profilin was coupled to a NHS-activated agarose matrix (NHS-activated HiTrap- column, Pharmacia Biotech, Inc., Piscataway, NJ) following the manufacturer's instructions. Chromatography steps were carried out at 4°C. Radiolabeled Mena protein was loaded onto the column in the presence or absence of 1 mg/ml GPPPPP (SEQ ID NO. 13) . Proteins were eluted from the column with buffer B (50 mM Tris-HCl (pH 7.2), 1 mM DTE, 5 mM MgCl 2 , 150 mM NaCI, 0.1% BSA) with 5 mg/ml poly-L-proline (1-10 kD, Sigma, St. Louis, MO) .
  • buffer B 50 mM Tris-HCl (pH 7.2), 1 mM DTE, 5 mM MgCl 2 , 150 mM NaCI, 0.1% BSA
  • the eluates were analyzed by SDS-PAGE and autoradiography. Autoradiography demonstrated that 83% of the input Mena was retained on the profilin-SEPHAROSE column as determined by phosphoimager analysis of the gel. Equal binding of the phospho- and dephospho forms of Mena was observed. In the presence of the competitor peptide, the amount of Mena retained was reduced to 13%.
  • Mena-profilin binding was also assessed by incubating 5 x IO 15 M of Mena with increasing concentrations of profilin to determine the % of Mena bound.
  • the profilin-SEPHAROSE matrix was diluted with Sepharose to create columns bound with known, increasing profilin concentrations.
  • the 5 x 10 15 M of in vi tro translated, radiolabeled Mena was mixed with profilin-Sepharose matrix and unbound Mena was washed from the column.
  • the bound Mena was quantified by scintillation analysis. This analysis demonstrated that the Mena binding is saturable.
  • Gst-AblSH3, GstScrSH3 (described by Gertler et al . , ibid., 1995; which is incorporated herein by reference in its entirety) .
  • the Gst fusions proteins were each bound to glutathione-SEPHAROSE as described previously.
  • the SH3-SEPHAROSE matrix was then gel electrophoresis and autoradiography. Gst alone was used as a control, and as expected, no binding to Gst was observed.
  • the AblSH3 retained 21 % and 14% of the input dephospho-, or phosphorylated input Mena, respectively.
  • the SrcSH3 retained 8% and 3% of the dephospho-, or phosphorylated Mena, respectively. In both cases, the dephosphorylated form of Mena was more efficiently purified from the lysate.
  • the Gst-SH3 fusion proteins were also used to purify Mena from RIPA head lysates.
  • the Gst-fusion proteins were each bound to the glutathione-SEPHAROSE matrix as described above.
  • RIPA head lysates were added to the matrix.
  • the matrix was washed, and the bound protein was eluted and analyzed.
  • both the broadly distributed and neural Mena forms were purified.
  • Mena Localization in Listeria Infection To determine if Mena is involved in the microfilament assembly required for Listeria motility, the distribution of Mena was examined in infected cells.
  • L. monocytogenes serotype l/2a EGD were grown in brain-heart infusion (BHI) broth (DIFCO Laboratories, Inc., Detroit, MI) at 37 °C with aeration.
  • BHI brain-heart infusion
  • PtK 2 cells were grown on coverslips, and bacteria from an overnight culture were added directly to the culture medium at a dilution of 1:200. After 1-2 hours the plates were extensively washed with fresh medium containing 25 ⁇ g/ml gentamicin to kill extracellular bacteria.
  • Knock-out mice in which the murine Mena coding sequence was replaced with the ⁇ -galactosidase gene and the neomycin resistance gene (neo) were generated i) to assess the consequences of eliminating the murine Mena protein during mouse development, ii) to permit examination of the expression pattern of Mena in embryonic mice, iii) to generate Mena " cell lines, and iv) to cross the mice with mice carrying oncogenes to study the effects of such double mutants.
  • Genomic Mena sequences used for these knock-out mice were obtained from the 129/Sv mice so that the homologous recombination could take place in a congenic background in 129/Sv mouse embryonic stem cells.
  • Mena genomic clones were isolated from a genomic library prepared from 129/Sv mice (Zhuang et al . , Cell 79:875-884, 1994; which is incorporated herein by reference in its entirety) using a random-primed Mena cDNA probe.
  • Plasmid pSAj8Geolox2DTA contains the 0-galactosidase/neomycin resistance gene fusion ( ⁇ Geo) and the Diptheria toxin gene under the control of the PGK promoter in a pBLUESCRIPT (Stratagene Cloning Systems, La Jolla, CA) vector backbone was used to create the targeting construct.
  • ⁇ Geo 0-galactosidase/neomycin resistance gene fusion
  • pBLUESCRIPT Stratagene Cloning Systems, La Jolla, CA
  • a 10 kb 5' Mena genomic fragment, the 4 kb cytoplasmic ⁇ Geo gene and the 1 kb fragment containing the genomic 3 ' untranslated sequence of Mena was inserted 5 ' of the Diptheria toxin expression cassette in the pSA/3Geolox2DTA vector backbone.
  • the targeting construct was linearized at a unique restriction site in the vector backbone and was transfected by electroporation into mouse embryonic stem (ES) cells.
  • ES mouse embryonic stem
  • 129/Sv derived ES cell line AK-7 described by Zhuang et al . (ibid.) was used for electroporation.
  • ES cells were routinely cultured on mitomycin C-treated (Sigma) SNL 76/7 cells (feeder cells) as described by McMahon and Bradley ( Cell 62:1073-1085, 1990; which is incorporated herein by reference in its entirety) in culture medium containing high glucose DMEM supplemented with 15% fetal bovine serum (Hyclone) and 0.1 ⁇ M (micromolar) ⁇ -mercaptoethanol .
  • the targeting construct for transfection 25 g (micrograms) of the targeting construct was linearized by digestion with Not I, phenol-chloroform extracted, and ethanol precipitated. The linearized vector was then electroporated into 1-2 x IO 7 AK-7 (ES) cells. The electroporated cells were seeded onto three 10-cm plates with one plate receiving 50% of the electroporated cells and the remaining two plates each receiving 25% of the electroporated cells. After 24 hours, G418 was added to each of the plates to a final concentration of 300 ⁇ g/ml (micrograms per milliliter) . The culture medium for each plate was changed every day for the first few days, and then changed as needed after selection had occurred.
  • ES x IO 7 AK-7
  • each positive clone was thawed and passaged once on feeder cells.
  • the transfected cells were trypsinized into single cells, and blastocysts obtained from C57BL/6J mice were injected with approximately 15 cells.
  • the injected blastocysts were then implanted into pseudopregnant mice (C57BL/6J x CBA) .
  • Six male chimeras arose from the injected blastocysts. All of the male chimeras gave germ-line transmission at a high rate as determined by the frequency of agouti coat color transmission to their offspring (Fl) in a cross with C57BL/6J female mice.
  • genomic DNA prepared from tail biopsies was digested with Eco Rl and probed with the a 3 kb 5 ' genomic sequence flanking the targeting construct. This probe detects a 16 kb fragment from the wild-type allele and a 10 kb fragment from the mutant allele. Therefore, a Southern analysis would show a single 16 kb band for a wild-type mouse, 16 kb and 10 kb fragments for a heterozygous mouse, and a single 10 kb band for a homozygous mutant mouse.
  • the resulting offspring (Fl) heterozygous (+/-) mice, were mated with sibling heterozygous mice to give rise to the homozygous (-/-) mutant mice.
  • chimeric mice or Fl heterozygous progeny from the chimera x C57B/6J mating were crossed with C57B/6J. Litters resulting from these crosses were harvested from pregnant females and stained for ⁇ -galactosidase activity. The embryos were dissected away from all the extra-embryonic tissue and the yolk sac was reserved for DNA analysis.
  • the embryos were fixed for one hour in a Fix solution containing (0.1 M phosphate buffer containing 0.2% glutaraldehyde, 2% formaldehyde, 5 mM EGTA (pH 7.3), 2 mM MgCl 2 ) .
  • the fixing solution was removed by three thirty-minute rinses with rinse solution (0.1 M phosphate buffer (pH 7.3) containing 2 mM MgCl 2 , 0.1% sodium deoxycholate, 0.2% NP-40) .
  • the fixed embryos were stained overnight in the dark in rinse solution containing 1 mg/ml X-gal, 5 mM sodium ferricyanide, 5 mM sodium ferrocyanide.
  • the embryos were rinsed with PBS and stored in the Fix solution before preparation for examination. Examination of stained tissue from fetal and postnatal mice heterozygous for the mutation demonstrated Mena expression pattern in the nervous system, somites, muscle tissue, neural crest cells and in the gut.
  • IMMORTOMOUSE is a mouse carrying a H-2Kb-tsA58 SV40 large T antigen transgene.
  • the progeny of the cross were subjected to PCR analysis as generally described above to identify progeny carrying the transgene and are heterozygous for the Mena knockout gene.
  • the progeny carrying both the transgene and the Mena knockout gene were back-crossed to the Mena ' heterozygotes.
  • the progeny of the back-cross were subjected to PCR analysis to identify homozygous Mena ' progeny carrying the transgene.
  • Cells from the resulting mice may be immortalized by culturing the cells at 33 °C in the presence of interferon.
  • ORGANISM Mus musculus
  • Ala Ala Ala lie Ala Gly Ala Lys Leu Arg Lys Val Ser Arg Val Glu
  • Arg lie Ala Glu Lys Gly Ser Thr lie Glu Thr Glu Gin Lys Glu Asp 430 435 440
  • Val Gly Arg Lys lie Gin Asp His Gin Val Val He Asn Cys Ala He 50 55 60
  • ORGANISM Mus musculus
  • ACAGACTTTC CACCAATGGA GGGATGCTAG ACAGGTGTAT GGTCTCAACT TTGGCAGCAA 420
  • ORGANISM Mus musculus

Abstract

La présente invention concerne deux nouveaux gènes de mammifères Mammalian Ena (Mena) et Ena-VASP like (Evl) codant de nouvelles protéines, Mena et Evl. Les protéines Mena et Evl comportent un domaine fonctionnel EVH1 discret responsable de la liaison du Mena avec l'ActA du Listeria et avec les protéines de cytosquelette de la zyxine et de la vinculine. Le domaine EVH1 du Mena est également responsable du ciblage, et suffisant au ciblage, de la localisation du Mena et des protéines hybrides basées sur le Mena sur des adhésions focales et sur la surface des Listeria, sur le site polaire où est induite la formation empanachée de l'actine. On décrit plusieurs procédés et compositions basés sur les gènes et protéines Mena et Evl de la présente invention permettant de cribler, d'isoler et de caractériser des facteurs endogènes et exogènes, des médicaments et des agents thérapeutiques utiles pour l'évaluation et/ou la régulation des phénomènes dynamiques du cytosquelette intervenant dans la morphologie, l'adhésion, la motilité, la croissance et/ou la différenciation cellulaires normales et anormales.
PCT/US1997/011669 1996-07-05 1997-07-03 Procede de criblage de proteines mena intervenant dans la dynamique des microfilaments WO1998001755A1 (fr)

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FR2806805A1 (fr) * 2000-03-22 2001-09-28 Centre Nat Rech Scient SEQUENCE PEPTIDIQUES COMPRENANT UN OU PLUSIEURS MOTIFS DE LIAISON AUX PROTEINES DE LA FAMILLE Ena/VASP, ET LEURS UTILISATIONS
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US20020037286A1 (en) * 2000-04-03 2002-03-28 Matthias Krause Methods for altering T cell and macrophage activation
WO2001074853A2 (fr) * 2000-04-03 2001-10-11 Massachusetts Institute Of Technology Methodes et produits permettant de reguler la motilite cellulaire
DE10058596A1 (de) * 2000-11-25 2002-06-06 Aventis Pharma Gmbh Verfahren zum Screening von chemischen Verbindungen zur Modulierung der Wechselwirkung einer EVH1-Domäne oder eines Proteins mit einer EVH1-Domäne mit einer EVH1-Bindedomäne oder einem Protein mit einer EVH1-Bindedomäne sowie ein Verfahren zum Nachweis besagter Wechselwirkung
CA2676179C (fr) 2007-02-02 2019-07-16 Albert Einstein College Of Medicine Of Yeshiva University Variantes d'epissages specifiques metastatiques de mena, et utilisation de celles-ci dans le diagnostic, le pronostic et le traitement de tumeurs
WO2011093989A1 (fr) * 2010-01-27 2011-08-04 Albert Einstein College Of Medicine Of Yeshiva University Méthodes de détermination d'agents ciblant les isoformes de mena et utilisations associées pour le diagnostic et le traitement de tumeurs métastatiques

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